It is increasingly evident that the transition from initiation to transcript elongation by RNA polymerase II is a major regulatory checkpoint in eukaryotic gene expression. During the current support period our laboratory has made significant advances in characterizing this transition. We have devised a working model for promoter clearance, the initial step in this process. Our clearance model includes the novel concept of an important role for transcript initiation factors. We have also demonstrated that a distinct postclearance transition occurs from 25-35 bases downstream of transcription start. Our analysis of the ability of RNA polymerase II to pass through the postclearance stage has suggested new insights into mechanisms through which polymerase can be trapped at this point in transcription. In the current application, we propose four specific aims, through which we will (i) test our initial models for the clearance and postclearance transitions (ii) determine the roles of individual transcription factors in these transitions, and (iii)identify underlying DMA sequences which have important effects on these transitions. Relevance to Public Health: Many genes whose correct expression is essential for human health are controlled through the extended pausing of the transcriptional machinery at a point just after the transcription of genetic information has initiated. It is not currently known how these "regulated gates" operate. In order to ultimately manipulate these gates for the benefit of human health, it is essential to uncover the molecular mechanisms involved. The research proposed here is aimed at providing this knowledge. The clearance and postclearance transitions which we study are test tube models for the regulatory points observed in the cell.